The impact of developmental stress on the functioning and vulnerability of CNS neurons

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The impact of developmental stress on the functioning and vulnerability of CNS neurons

Pienaar, Ilse-Sanet

2008-12

The overall objective of this thesis is to provide additional data to assist clinicians and
experimental neurologists alike in the quest for better understanding, more accurately
diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD).
The general theme of the thesis is the interaction between certain environmental stimuli,
including the exposure to adverse events during early central nervous system (CNS)
development and the manifestation of elements of neurodegeneration, whether by means of
neurochemical changes or expressed as a dysfunctional voluntary motor system.
The first chapter provides a general introduction to the research theme of the thesis. This
includes, in particular, a discussion on current understanding concerning the etiology and
clinical profile of PD, the relative contribution made by genetic factors compared to
environmental ones, and current treatment strategies for treating the disease. Mention is also
made of the failure of these therapeutic applications for reversing or protecting against the
disease, due to the side-effects associated with them. The material covered in chapter 1
provides the basis for the more complete discussion concerning these various aspects,
contained in the chapters to follow.
The overall aim was also to characterise the effects of commonly used toxin-induced animal
models of PD, and the extent of vulnerability that the CNS displays towards them. The
destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points
along the nigrostriatal tract is used extensively to model PD pathology in rats and is an
established animal model of the disease. However, mature or even aged animals are mainly
used in these studies, while the effects that the toxin might have on the developing CNS remain
unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the
young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a
unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial
forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a
direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum.
Although both lesion types were used, the MFB model is considered a more accurate portrayal
of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of
the disease. The different lesions’ effects on motor function were determined by observing
animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of
a cylindrical enclosure. Following the final behavioral assessment, the concentration of
dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using
4
HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The
HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the
lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment,
there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic
terminals that may be sufficient for maintaining relatively high extracellular and synaptic
concentrations of DA. However, since substantial changes in motor-function were observed, it is
suggested that the capacity of the remaining dopaminergic neurons to respond to increased
functional demands may be limited. In addition, the behavioral results indicate that the distinct
indices relating to different functional deficits depend on the lesioning of anatomically distinct
structures along the nigrostrial tract.
It has long been known that far fewer women are diagnosed with PD than men are. This
seeming protection offered to females against degenerative disease of the CNS may relate to
estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly
defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United
States alone, the aim of chapter 2 was to examine the evidence for a possible relationship
between PD and the female reproductive hormone estrogen. A review of the current literature
available on the topic was performed by consulting Medline, and by performing a search of the
case-reports contained within the World Health Organization’s (WHO) International Drug
Monitoring database, for possible PD-related symptoms that may arise from estrogen
replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen
protects women from obtaining the disease, or at least some features of it. Intensive research
efforts are called for, with sufficient power to establish the relationship between ERT and the
onset and development of parkinsonism. Chapter 3 reports on the results obtained from an
experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher
dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14
days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6-
OHDA the female rats were less impaired than males in making adjustment steps in response
to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine
hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender
differences in cell survival factors and/or other promoters of neuroplasticity may have
contributed to the beneficial outcome seen in the females. For example, nerve growth factor
(NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It
is unclear whether gonadal steroids are involved, and, if so, whether female hormones are
protective or whether male hormones are prodegenerative. Determining the mechanisms for the
improved outcome seen in the young female rats may lead to potential treatment strategies
against PD.
5
Many studies have shown that early life stress may lead to impaired brain development, and
may be a risk factor for developing psychiatric diseases, including clinical depression. However,
few studies have investigated the impact that early stress may have on the onset and
development of neurodegenerative disorders such as PD. The study reported on in chapter 5
conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well
characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6-
OHDA. The combined effects of these models on motor deficits and brain protein levels were
investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion
with a battery of tests that are sensitive to the degree of DA loss sustained. The results
show that animals that had been subjected to MS display poorer performance in the vibrissae
and single-limb akinesia test compared to non-MS control animals (that had also been
subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH
staining in MS rats compared to non-MS ones. The results from this study therefore suggest
that exposure to adverse experiences during the early stages of life may contribute towards
making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring
during mature stages of life. Therefore, taken together, early exposure to stress may predispose
an individual towards the onset and development of neurodegenerative disease, which
especially becomes a threat during the later stages of adult life.
Moreover, within the framework of these characteristics, the capacity of a widely-used
pharmacological agent (statins) was tested for possible future therapeutic application in PD
(chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests
that it may associate with defective activity of complex I of the mitochondrial electron transport
chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased
oxygen free radical production, depressed antioxidant enzyme activities, and greater
susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for
lowering cholesterol levels in patients who display elevated concentrations of low-density
lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on
motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I
inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with
simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor
function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb
placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally
lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the
vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified
concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2-
6
Dimensional electrophoresis (2-DE) with subsequent identification of the spots using
electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was
performed and the results BLAST-searched using bio-informatics tools for naming the identified
peptides. The motor test results indicate that while unilateral rotenone causes behavioral
asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced
ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in
protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly
classified according to their cellular function into 6 categories, with the majority involved in
energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated
techniques and non-biased ability to quantify proteins, provides a methodology with which to
study the changes in neurons associated with neurodegeneration. As an emerging tool for
establishing disease-associated protein profiles, it also generates a greater understanding as to
how these proteins interact and undergo post-translational modifications. Furthermore, due to
the advances made in bioInformatics, insight is created concerning their functional
characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss
major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped
that the application of this technology will lead towards a presymptomatic diagnosis of PD, and
the identification of risk factors and new therapeutic targets at which pharmacological
intervention can be aimed.
The final chapter (chapter 8) provides a retrospective look at the academic work that had
been performed for the purpose of this thesis, recaps on the main findings, and also highlights
certain aspects of the project and provides relevant suggestions for future research. Lastly, the
appendix provides a detailed overview of the methods followed for the experiments described in
this thesis. It provides not only a comprehensive description of the techniques that had been
followed, but provides information concerning the care taken with the animals (i.e. post-surgery)
in order to control for the potential influence of experimental variables on the results.